Use of antibodies against tumor-associated antigen

ABSTRACT

The invention relates to the use of a preparation based on an antibody directed against a tumor-associated antigen, for producing a medicament for the intra-operative treatment of tumor patients, in particular for the prophylactic treatment to prevent the dissemination of tumor cells. The invention further relates to a kit for the intra-operative treatment of tumor patients, comprising the medicament and means for diagnostically determining malignant tumor cells.

The invention relates to a new use of an antibody preparation as well asto a kit for the intra-operative treatment of tumor patients.

Tumors form due to the unchecked cell growth which leads to theformation of solid cell agglomerates in case of epithelial cells. Incase of benign tumor tissue, it is assumed that the cell growth islimited and secondary tumors or metastases will not occur. In cancerdiseases, however, malignant tumors form, and in the progressing stagesecondary tumors and metastases occur. Most frequently, cancer formswith epithelial tumors occur which inter alia concern breast, stomach,intestines, pancreas, lungs, prostate and ovaries.

Cancer is a wide-spread disease and is lethal in many cases. The therapyof cancer usually comprises the removal of a solid tumor, and a furthertreatment which is to prevent and reduce, respectively, metastases.Besides surgery, the standard therapies include chemotherapy andradiation therapy. Despite the comprehensive therapy which ofteninvolves severe side effects, the success of treatment is insufficient.The relapse rate in intestinal cancer is approximately 45%. Metastaticepithelial cancer is considered to be nearly incurable. Therefore, inthe treatment of cancer patients it is important to prevent, and reduce,respectively, the formation of metastases.

Tumor cells are capable of disseminating from primary tumors in bodyliquids and other organs. These disseminated tumor cells may be in theirdormant state and often cannot be attacked by a chemotherapy(radiotherapy). Such a treated patient seems to be in a cured state,which is described as “minimal residual disease”. Dormant tumor cells,however, have a potential of forming metastases if they become growingand metastasizing cells.

Immunotherapy constitutes an innovative possible treatment of cancerpatients. Both active and also passive immunotherapy are acknowledgedmeasures for supporting the immune system.

The adaptive immune system of humans consists of two essentialcomponents, the humoral and the cellular immunity. The adaptive immuneresponse partially is based on the clonal selection of B- andT-lymphocytes and in principle allows for the recognition of any desiredantigen as well as for the build-up of an immunological memory. Thesecharacteristics of the adaptive immune system are generally usefullyaddressed in vaccinations.

Each B-cell produces an antibody with a defined binding specificity.This antibody is also present as a specific receptor in the membrane ofthe B-cell producing it. The humoral immune response against antigensrecognized as foreign is based on the selective activation of thoseB-cells which produce such antibodies that can bind to an epitope of therespective antigen. For the antibody diversity, DNA rearrangements inthe course of B-cell differentiation play a decisive role.

There are several possible ways of interfering in the immune system.

1. Passive Antibody Therapy:

For therapeutic purposes, it is possible to supply to an organismantibodies required for a certain function within this organism. Thistype of application is called passive immunotherapy, and it can be usedin various medical indications, e.g. in the immunotherapy of cancer(Immunol. Today (2000), 21:403), intoxications (Toxicon (1998), 36:823;Therapie (1994), 49:41) and infections (Clin. Infect. Dis. (1995),21:150). In these cases, antibodies can be used which either have beenderived from appropriately immunized animals or can be recovered fromcells by various biological or molecular-biological techniques (e.g.hybridoma technique, phage-display technique, etc.) via theimmortalization of immunoglobulin genes.

2. Active Immunization:

To modulate the immune system, an immunization with antigens can beused. Antigens are molecules, molecule complexes or whole organisms towhich antibodies can bind. Not all the antigens induce an immuneresponse, i.e. not all the antigens are immunogenic. Certain smallmolecules are not registered by the immune system (haptens), suchsmaller molecules can be presented to the immune system in suitableform, and thus be made immunogenic. Such a method is the coupling of thehapten to an immunogenic molecule, a so-called carrier molecule. For anactive immunization, also antibody preparations can be used, such asdescribed in EP 1140168.

Tumor cells can be attacked by the immune system only to a limitedextent, since they are hardly different from normal cells and specificantibodies therefore are missing. Much research is directed to theidentification of suitable targets, i.e. target antigens, for thepreparation of tumor-specific antibodies. The immunotherapy for thetreatment of cancer then either comprises the passive therapy by thedirect administration of the specific antibodies, or the activevaccination with suitable antigen-targets for stimulating the immunesystem and generating the specific antibodies in vivo.

One approach of relatively specifically destroying tumor cells is thepassive immunotherapy with antibodies directed against tumor-associatedantigens (TAA) (Immunology Today (2000), 21:403-410; Curr. Opin.Immunol. (1997), 9:717). Another approach of destroying tumor cells isan active vaccination which triggers an immune response against TAA.This immune response thus is also directed against the correspondingtumor cells (Ann. Med. (1999), 31:66; Immunobiol. (1999), 201:1).

Certain TAAs are defined as relevant “targets” for the development ofimmunotherapeutic agents for the prophylaxis and/or treatment of cancer.TAAs are structures which preferably are expressed on the cell membraneof tumor cells, thereby allow for a differentiation relative tonon-malignant tissue, and thus can be viewed as targets for thediagnostic and therapeutic applications of specific antibodies.

In the course of the discovery and the subsequent characterization ofvarious TAAs it has been found that they often have important functionsfor cancer cells. They allow the degenerate cells to have propertiescharacteristic of the malignant phenotype, such as, e.g., an increasedadhesion capacity, or an increased uptake of growth factors, which arehighly important for establishing metastases. However, in certainstages, such antigens may very well also be expressed on normal cellswhere they are responsible for normal functions of these cells. Anexample of this is the Lewis Y carbohydrate antigen which appears on theplurality of tumors of epithelial origin, but also plays an importantrole during the fetal development of epithelial tissues. It has beenshown that the expression of this antigen in lung cancer is associatedwith an unfavorable prognosis, since Lewis Y positive cancer cellsapparently have a higher metastatic potential (N. Engl. J. Med. 327(1992), 14).

In EP 0 528 767, the use of a humanized anti-Lewis Y antibody for thetreatment of epithelial cancer has been described.

Among the further known tumor-associated carbohydrate structures, thereare, e.g., all those Lewis antigens which are highly expressed in manytypes of epithelial cancers. Among them are Lewis x-, Lewis b- and Lewisy-structures, as well as sialylated Lewis x-structures. Othercarbohydrate antigens are Globo H-structures, KH1, Tn antigen, TFantigen, the alpha-1,3-galactosyl epitope (Elektrophoresis (1999),20:362; Curr. Pharmaceutical Design (2000), 6:485, Neoplasma (1996),43:285).

Other TAAs are proteins which are particularly highly expressed bycancer cells, such as, e.g. CEA, TAG-72, MUC1, Folate Binding ProteinA-33, CA125, EpCAM, HER-2/neu, PSA, MART, etc. (Sem. Cancer Biol.(1995), 6:321). Relevant TAAs often are surface antigens of epithelialcells which occur in larger numbers in growing cells, such as fetaltissue, and also in tumor tissue.

Direct therapeutic applications of antibodies against TAA are based onpassive immunotherapies, i.e., a specific antibody is systemicallyadministered in a suitable amount to cancer patients, and has animmunotherapeutic effect. The biological half-life of such agents willdepend on their structure and is limited. Therefore, it is necessary tocarry out repeated applications. When using xenogenic antibodies (e.g.murine monoclonal antibodies, MABs) however, this can lead to undesiredimmune reactions which may neutralize a possible therapeutic effect andmay cause dangerous side effects (anaphylactic reactions). Therefore,such immunotherapeutic agents can be administered for a limited timeonly.

A better tolerance is obtained by reducing the xenogenic structures ofthe antibody and by introducing human structures, e.g. with chimeric orhumanized antibodies. Also systems for producing specific humanantibodies are being developed. Thus, according to the prior art,certain cell lines, organisms or transgenic animals can produce humanantibodies.

Different publications deal with the risk of metastasis formation by theliberation of tumor cells as it may occur with a surgical treatment. Asdescribed in Clin. Cancer Res. 4(2), 343-8 (1998), particularly during aresection of a tumor, tumor cells can be determined in a blood sample. Adirect link is seen between the intra-operative dissemination ofhematogenic tumor cells and the “minimal residual disease” as well asthe formation of metastases. In Semin. Surg. Oncol. 20(4), 329-33 (2001)it has therefore been suggested to take additional perioperativemeasures of antibody therapy or cytotoxic therapy.

A combined treatment of tumor patients has been described in WO00/69460. Prior to a surgical treatment, patients at first are treatedfor 8-24 weeks with an antibody preparation combined with a cytotoxicagent, at weekly intervals, so as to reduce the tumor size. After thetumor resection has been effected, the patient again is subjected to thecombined immunotherapeutic treatment. This perioperative treatment atfirst comprises a complete treatment cycle weeks before surgery, andlater on, a further cycle weeks after the surgery. In this document,antibodies are administered which lead to the cell death via an ADCC ora CDC function (cf. page 5, lines 10-17). The thus treated patientsexhibit an improved survival rate as well as a reduced spreading of thetumor (TTP).

According to US 20010006618, antibody preparations have been suggestedfor diagnostic use during a surgical, intravascular, laparoscopic orendoscopic intervention. Intra-operative lesions are to be detected byusing a labelled antibody preparation. In doing so, the labelledantibody is administered within 48 hours prior to the intervention, andafter the surgery, the patient is examined with a view to the possibledissemination of the labelled antibody. In addition to its use fordetecting the tumor, the labelling substance may also be used fortreating the tumor, e.g. by using a photoactive reagent as labellingsubstance which after binding the antibody, can be activated by theaction of light.

According to U.S. Pat. No. 4,643,971, a selection of tumor-specificantibodies has been suggested for determining biliary cancer prior to arespective surgery.

WO 01/23005 relates to the administration of antibodies which areconjugated with a dye so as to illustrated the tumor rim. The antibodydye conjugates preferably become enriched in the rim region of the celltissue of a tumor and thus allow for an optical imaging of the rimregion (cf. p. 8, 1^(st) complete paragraph).

The invention has as its object to improve the treatment of cancerpatients in so far as the dissemination of tumor cells and the formationof metastases, respectively, are largely suppressed.

According to the invention, this object is achieved by the subjectmatter of the claims.

The use according to the invention relates to an antibody-basedpreparation for the production of a medicament, which antibody isdirected against a TAA. This medicament is administered within the scopeof surgical interventions for the intra-operative treatment of tumorpatients, wherein tumor cells are immunocomplexed. The immunocomplexformation occurs during surgery, i.e. primarily on tumor tissue or cellswhich become intra-operatively accessible.

The intra-operative treatment is the prerequisite for the functionalactivation of the immune system against the tumor cell, whereupon thelatter is immediately lysed. The dissemination of tumor cells which mayoccur during certain surgical procedures is then largely prevented. Thefact that a dissemination of tumor cells may even occur during thebiopsy, i.e. during a relatively slight invasive surgical intervention,has, e.g., been shown in female breast cancer patients. There, proof ofdisseminated tumor cells was not possible before surgery, after thebiopsy, the level of circulating epithelial cells which correspond todisseminated tumor cells, increased highly in some instances.

According to the invention, the use of the medicament is primarily aprophylactic one so as to prevent the dissemination of tumor cells insecondary organs or in bone marrow.

The treatment according to the invention comprises both prophylactic andalso therapeutic measures. The treatment does not only serve humanmedicine, it may also be indicated in surgical interventions on variousmammals.

The term “intra-operative treatment” according to the invention isintended to mean the treatment of tumor patients which ensures a highantibody titer for the duration of a surgery. Practically, the antibodypreparation is administered only a few hours before, i.e. immediatelywhile the patient is being prepared for the surgery, or during thesurgery itself. The treatment usually is carried out within 24 hoursprior to surgery, preferably within 8 hours, more preferred within 4hours, most preferred within one hour before the surgical intervention.The intra-operative treatment according to the invention thus differsfrom a perioperative treatment of the prior art which provides for arepeated treatment of the patient over several weeks prior to surgery,as well as a further treatment period after surgery.

According to the invention, surgical interventions are provided for thepartial or complete tumor resection of solid tumors. Tumor tissuepreferably is removed with a no-touch surgical technique so as to reducethe risk of the dissemination of tumor cells into the periphery rightfrom the beginning.

Immediately before and during the surgical intervention, according tothe invention the tumor patient usually will receive a single dose of anantibody preparation so as to achieve a certain serum content. Startingfrom a specific immunoglobulin content of approximately 10, preferablyat least 50, most preferred more than 100 μg/ml serum, a protectionagainst a possible spreading of tumor cells into the circulatory systemis thus built up. After an intravenous treatment, the antibody titer ispractically immediately available. Further systemic treatments comprisethe intramuscular, subcutaneous, intradermal or mucosal, such as oral ornasal administration. In doing so, a delayed availability must becounted on. The antibody preparation can also be locally administered,i.e. to the tumor tissue and/or in the wound area after removal of thetumor. Various modes of administration may appropriately be combined,e.g. an i.v. infusion shortly before surgery, as well as a local dosedirectly applied into the wound region. Practical application means forthe localized administration comprise, e.g., ready-to-use applicatingmeans, such as catheters, syringes or sprays, suitable for distributingliquid medicaments.

Particularly in endoscopic or microinvasive surgical methods, the localapplication is advantageous. Thus, during biopsy, the medicamentpreferably is introduced via the puncture channel, or in parallel, viaappropriate syringes or catheters, respectively.

Small tumors or a sample of a tumor tissue are usually removed bybiopsy. On the basis of the biopsy material, the pathologist willdiagnose whether the tissue is from a benign or a malignant tumor.Patients who are subjected to a biopsy as such are not yet subjected toa cancer therapy. According to the prior art, the patient will receiverespective medicaments only after a cancer disease has been positivelydiagnosed. According to the invention, however, a high-risk patient willalready receive the appropriate prophylactic treatment so as to excludea dissemination of tumor cells due to the biopsy itself.

The medicament used according to the invention as such is well tolerableand usually is administered to the tumor patient only once. Theprophylaxis therefore is also indicated if the risk of a cancer diseasehas not yet been confirmed, and if the surgical intervention will becarried out to decide on the malignancy of a tumor. Besides the usualdiagnostics by the pathological finding, according to the inventionthere exists the further possibility to determine the immune complexesformed and to obtain in this manner an indicator for the malignancy ofthe tumor. A prerequisite for this is that the antibody used is directedagainst a TAA which distinguishes benign from malignant tumor tissue.

In case of a cancer disease, according to the invention immune complexeswith tumor tissue, or with tumor cells, respectively, are obtained whichare determinable not only at the tumor tissue, following resection ofthe tumor, but also in peripheral body fluids. After the treatment,blood or serum samples of the patient can be qualitatively and/orquantitatively assayed for immune complexes. The analysis methods areper se common analysis methods with fractionation and enrichment of theimmune complexes and/or immunoreaction with a specific antibody, such asone directed against the Fc portion of the antibody employed for thetreatment. If the antibody used comprises murine structures, the formercan also be bound with an anti-murine antibody, as defined by a capturestep. The detection of the binding usually is effected with anappropriate antibody labelling, such as a fluorescence label, or with acolor reaction.

According to the invention, furthermore, a kit for intra-operativetreatment of tumor patients is provided, which kit comprises

-   a) a medicament based on an antibody, directed against a    tumor-associated antigen, and-   b) a means for the diagnostic determination of malignant tumor cells    which are immunocomplexed with the antibody.    The antibody of the kit preferably is a native, i.e. a functionally    active, antibody. This antibody thus preferably has not an adhered    label or other detection agent so as not to impair its    functionality.

The inventive use preferably is effected with antibodies directedagainst an epitope of a surface antigen of a tumor cell. Optionally, aTAA from cells of solid tumors, or a panel of TAAs is chosen, inparticular individual TAAs of the tumor of the respective patient.Patients having primary tumors can be treated just as well as patientswith secondary tumors.

As a rule it must be assumed that by an antigen which imitates aproteinaceous epitope of a TAA, a polypeptide of at least five aminoacids is to be understood.

Among the epitopes of the inventively used antibody, preferably there isat least one epitope of a human antigen, selected from the group of thepeptides or proteins, in particular EpCAM, NCAM, CEA and T cell peptideswhich preferably are derived from tumor-associated antigens,furthermore, the carbohydrates, in particular Lewis Y, Sialyl-Tn, GloboH, and the glycolipids, in particular GD2, GD3 and GM2. Preferredepitopes are derived from antigens which are specific for epithelialtumors and, e.g., occur increasedly in breast cancer, cancer of thestomach and intestines, of the prostate, pancreas, the ovaries and thelungs. Among the preferred epitopes are those which primarily trigger ahumoral immune response, i.e. a specific antibody formation in vivo.Preferably, the immunogenic antibody according to the invention can alsotrigger a T-cell specific immune response, whereby, as a reaction to theadministration of the antibody, antibodies not only of the IgM class,e.g., are formed, but also of those of the IgG-class.

As an alternative, especially those antigens can be selected as epitopesas defined by the invention, which generate a T cell specific immuneresponse. Among them are primarily also intra-cellular structures or Tcell peptides, respectively. The intra-operative treatment according tothe invention may, e.g., also be used to reduce the volume of a pleuraleffusion, i.e. an accumulation of fluid in the pleural cavity. Pleuraleffusions often occur in patients who suffer from epithelial cancer.

Further preferred proteinaceous epitopes which are particularlyexpressed on cancer cells of solid tumors are, e.g., TAG-72, MUC1,Folate Binding Protein A-33, CA125, HER-2/neu, EGF receptors, PSA, MART,etc. (cf. e.g., Sem. Cancer Biol. 6 (1995), 321). Moreover, alsoso-called T cell epitope peptides (Cancer Metastasis Rev. 18 (1999),143; Curr. Opin. Biotechnol. 8 (1997), 442; Curr. Opin. Immunol. 8(1996), 651) can be used.

Suitable epitopes are expressed at least in 20%, preferably at least in30% of the cases of tumor cells of a certain cancer type, furtherpreferred in at least 40%, in particular in at least 50% of thepatients.

Carbohydrate epitopes preferred according to the invention aretumor-associated carbohydrate structures, such as the Lewis antigens,e.g. Lewis x-, Lewis b- and Lewis y-structures, as well as sialylatedLewis x-structures. Furthermore, also Globo H-structures, KH1, Tnantigen, TF antigen, the alpha-1,3-galactosyl epitope are preferredcarbohydrate antigen structures within the scope of the presentinvention.

A particularly good target for the inventively used antibody is theLewis Y antigen. Particularly preferred is a humanized antibody, such asdescribed in EP 0 528 767. This antibody recognizes Lewis Y antigen ontumor cells, or on surface receptors of tumor cells, respectively.

It has been found that the surface receptors of a tumor cell with anaberrant glycosylation form relevant epitopes as defined by theinvention whereby this glycosylation can be functionally blocked byantibodies. This does not only apply to one certain surface receptor,such as the EGF receptor or Her-2/neu receptor. Practically all thetumor-specific receptors which are characterised by the aberrantglycosylation are simultaneously blocked. Among them are, e.g., all thereceptors of the EGF receptor family, the CD55 (791Tgp72/DAF-decayaccelerating factor) receptor, the transferrin receptor and theP-glycoprotein. Thus, the tumor cell is attacked on the basis ofdifferent mechanisms of action.

It has also been found that antibodies directed against an aberrantglycosylation bind to several receptors of the family of the EGFreceptors in a functional manner and that thus the signal cascade forthe induction of cell growth can effectively be blocked. In Austrianapplication A 995/2002 it could be shown that in particular the erk1 anderk2 isoforms of the MAP kinase can be functionally bound by theantibodies employed according to the invention. The binding of thegrowth factors to the receptors was thus prevented or reduced,respectively. Compared to the immunotherapy with antibodies against theproteinaceous extracellular part of the EGF receptor, this treatment ismore specific since the unusual tumor-associated carbohydrate structuresare missing on EGF receptors of normal cells. On the other hand, thetreatment is more universal, since simultaneously different receptorswith the same aberrant glycosylation are blocked.

By this use according to the invention, thus also the mitogenicstimulation of a cancer cell by EGF or heregulin is prevented. Thespecific binding of the antibody to a tumor-associated glycosylation ofcancer cells blocks the interaction of the receptors of growth factorswith their physiological ligands and inhibits the signal transductionthrough these receptors, and thus, the cell growth.

Simultaneously, such an antibody is capable of specifically attackingthe tumor cell on account of its action within the humoral and cellularimmune system. Tumor cells which express the EGF receptor, or receptorsof the EGF receptor family, respectively, according to the invention arespecifically bound and can be lysed.

Methods for locating suitable antigenic structures, modelling andpreparation of the TAA-derived peptides, polypeptides or proteins, or ofnucleic acids coding therefore, respectively, furthermore lipoproteins,glycolipids, carbohydrates or lipids are known to the person skilled inthe art and can be provided for the respective tumor-specific structurewithout undue experimental expenditures. Furthermore, the methods ofproducing the specific antibodies are known which are suitable accordingto the invention.

In a special embodiment, an antibody mixture of various antibodieshaving specificity for TAA, in particular for at least two equal ordifferent epitopes of an adhesion protein, such as a homophilic cellularmembrane protein, such as EpCAM, is used. Likewise, a combination ofantibodies having specificity for at least one TAA epitope of the Lewiscarbohydrate antigens, in particular Lewis Y, is preferred.

According to the invention, the relevant antibody is primarily employedfor passive immunization, and in particular is administered only once.Thus, no special side effects are expected, even if the inventiveantibody is derived from a non-human species, such as a murine antibody.However, it is expected that a recombinant, chimeric, such as ahumanized antibody combined with murine and human components, or a humanantibody will be particularly tolerable for the administration onhumans.

By the term “antibody”, antibodies of any type are to be understood, inparticular monospecific or polyspecific monoclonal antibodies, or alsochemically, biochemically or molecular-biologically produced antibodies,or polyclonal antibodies having a certain specificity, such as an immuneserum or a fraction of an immune serum.

The antibody used according to the invention usually is a nativeantibody which possibly has been isolated from an organism or patient.Native antibodies are hetero-tetrameric glycoproteins assembled of twoidentical light chains and two identical heavy chains.

Yet, also an antibody derivative may be used which preferably isselected from the group of antibody fragments, conjugates, homologues orderivatives, yet also complexes with additional effector functions. Inany event it is preferred for the antibody derivative to contain atleast parts of the Fab fragment, preferably together with at least partsof the F(ab′)₂ fragment, and/or parts of the hinge region and/or of theFc portion of a lambda or kappa antibody.

Furthermore, also a single-chain antibody derivative, such as aso-called single chain antibody having effector functions as defined bythe invention may be employed. The antibody according to the inventionpreferably is of the type of an immunoglobulin, such as an IgG, IgM, IgAor IgD.

According to the invention, the antibody binds directly to a tumor cellor to metastases or micrometastases. An thus formed immune complex ofthe antibody is the prerequisite for the humoral and cellular activitiesof the immune system, expressed by an antibody-dependent cellularcytotoxicity (ADCC) and/or a complement-dependent cytotoxicity (CDC)effector function. These effector functions are determined by means ofstandard tests.

High-affinity antibodies are preferred according to the invention. Inparticular, antibodies are used which bind with an affinitycorresponding to a dissociation constant of below a Kd value of 10⁻⁶mol/l, preferably less than 10⁻⁷ mol/l, most preferred 10⁻⁸ mol/l, orless.

A possible treatment objective is the effective binding and reduction oftumor cells so as to prevent their dissemination as far as possible.Simultaneously, also particularly the disseminated tumor cells areattacked. The number of tumor cells, or micrometastases, respectively,detectable in blood, bone marrow or organs is significantly reduced byprophylaxis and therapy according to the invention. The formation ofmetastases is to be retarded thereby, and their growth is to be at leastslowed down. Thus, by the immunotherapy according to the invention, therelapse-free life span and, thus, also the total survival time of thepatients can be increased. An indicator for the success of the treatmentis the significant reduction of tumor cells in blood, serum or bonemarrow.

The medicament used according to the invention advantageously isprovided in a suitable formulation. Preferred are such formulations witha pharmaceutically acceptable carrier. The latter comprises, e.g.,auxiliary substances, buffers, salts and preservatives. Preferably, aready-to-use infusion solution is provided.

Since an antibody is comparatively stable, medicaments based onantibodies or their derivatives have the substantial advantage that theycan be put on the market as storage-stable solutions or as a formulationin a ready-to-use form. The latter is preferably storage-stable atrefrigerating temperature up to room temperature. The medicament usedaccording to the invention may, however, also be provided in frozen orlyophilized form which may be thawed or reconstituted, respectively,upon demand.

The antibody solution provided may be administered intravenously as abolus injection or also in diluted form. The medicament may, e.g., beprepared as an infusion preparation in a 1:10 to 1:100-fold dilutionwith physiological saline solution.

To saturate the relevant surface antigens of the tumor cells, usually ahigh dose of at least 50 mg, preferably at least 100 mg, most preferredat least 200 mg is administered per patient. The maximum dose is limitedby the tolerability of the antibody and will depend on its specificityand avidity. Humanized antibodies and human antibodies, respectively,are the best tolerable. A dose of up to 1 g or in some cases of up to 2g per patient and treatment may very well be advantageous.

Usually the patient will be further examined after the surgicalintervention so as to possibly carry out a suitable cancer treatment.Should it turn out after the surgical intervention that the tumor hasthe relevant TAA, an immediate further treatment with the sameantibodies that had been used intra-operatively and/or withimmunotherapeutically active other antibodies may be carried out.

The usual treatment for passive immunotherapy comprises repeatedinfusions at regular intervals, such as weekly for a period of time offrom 6 to 24 weeks, at a dose ranging from 1 to 10 mg/kg, preferablyranging from 2 to 6 mg/kg. The treatment is preferably repeated atcertain time intervals, corresponding to the half-life of the antibodyused which usually is in the range of from 5 to 30 days. By a specialderivatization of the antibody it is possible to lengthen the half-lifeto up to several months, and to thereby lengthen the treatment intervalsaccordingly.

The concentration of the active substance of the medicament will dependon its tolerability. A particularly well tolerated preparation based ona humanized antibody can be administered directly to the patient in ahigh concentration and without being further diluted. By the preferredconcentration in the range of from 0.1% to 10%, preferably 1% to 5%, itis possible to keep low the administered volume and the respectiveinfusion time.

The combination with known adjuvant treatment methods is quite common.Among them are agents for radiotherapy or chemotherapy, such as themonotherapy or polytherapy. For reasons of the different mechanisms ofaction, the immunotherapy preferably is combined with thepolychemotherapy.

Agents preferably used for chemotherapy are alkylating pharmaceuticalpreparations. Thus, e.g., agents containing taxane, anthracyclines orplatinum are preferred. All the conventional preparations which areemployed for the various cancer treatments can be further combinedaccording to the invention. The chemotherapeutic agents usually areadministered intravenously or perorally. Peroral administration forms ofthe chemotherapeutic agents possibly can also be administered with aperoral form for the immunotherapy according to the invention as acombination preparation.

In the following, the preparation of a medicament produced according tothe invention as well as its intra-operative use are described by way ofexample. The following examples and figures shall further explain thepresent invention, but not restrict it.

FIG. 1 shows the inhibition of xenograft growth by IGN311 and ABL364after inoculation with 1×10⁶ A431 cells in mice.

FIG. 2 shows the inhibition of xenograft growth by IGN311 afterinoculation with 3×10⁶ A431 cells in mice. The treatment with theantibodies was carried out within one day after dissemination of thetumor cells.

FIG. 3 shows the tumor weigths without and with a treatment with IGN311and ABL364.

FIG. 4 shows the reduction of the circulating EpCAM positive cells inblood after treatment with a murine IgG2a Mab with Ep-CAM mimickingproperties.

EXAMPLES Example 1 Preparation of a Lewis Y Antibody IGN311

IGN311 is a humanized antibody derived from Br55-2 murine IgG3 antibody.This murine antibody is derived from the hybridoma cell line BR55-2(BR55-2/IgG3), deposited on Feb. 17, 1987, at the American Type CultureCollection, Rockville Md. 20852, USA, under ATCC HB 9324.

The humanized antibody was prepared by CDR grafting (Cancer Research 56,118-125, Mar. 1, 1996). As constant region of the heavy chain, humanIgG1 was employed. The humanization was effected according to EP 0 528767. The antibody was produced in SP2/0 cells.

The IGN311 obtained proved to be highly active: Human complement wasactivated with a CDC approximately 10-fold increased relative to themurine antibody so as to lyse Lewis Y-positive tumor cells. The activityof the IGN311 was tested against a selection of Lewis Y-positive humantumor cell lines in the presence of human complement (CDC) (SKBR5,breast cancer, SW948, colon cancer; SW2, small cell lung cancer; andMCF7, breast cancer). The ADCC was also tested against a certainselection of tumor cell lines. The binding affinity remainedapproximately equal also after humanization.

The product was formulated in phosphate-buffered saline solution andfilled into containers at a concentration of 10 mg/ml. Depending on itsrespective use, this stock solution will be diluted before being used.For the i.v. product, the solution is diluted 1:50 with physiologicalsaline solution. If the preparation is to be applied locally in thewound area, the preparation will be used without being diluted.

Application During Tumor Resection

Patients diagnosed with a cancer of the intestines are prepared for thesurgical removal of the tumor. Before the treatment, a blood sample istaken. Treatment with the i.v. product is effected immediately beforesurgery over a period of time of 2 hours. The dose administered was 50mg. Subsequently, the tumor is removed within 4 hours.

Blood samples are taken immediately after the induction of anaesthesia,after tumor resection and 24 hours after surgery. The tumor cells areenriched from the fresh blood samples, applied to slides and stored at−20° C. until immunocytochemical analysis.

A sample of the removed tumor tissue is prepared for theimmunohistochemical examination.

Tumor and Tumor Cell Determination

An immunohistochemical determination of Lewis Y antigen is recommendedfor the surgically removed tumor tissue. For the histologicaldetermination of the Lewis Y antigen, de-paraffinated tissue isincubated with the murine antibody BR55-2 and subsequently with abiotinylated goat anti-mouse IgG (Vector Laboratories). Non-specificbinding sites are blocked with normal goat serum. To determine theimmune complex, horseradish peroxidase streptavidin (1:325 inphosphate-buffered physiological saline solution) is added, and stainingis performed (cf. in this respect J. E. Beesley, chapter 2.5:Avidin-biotin methods in Immunocytochemistry, edited by Oxford Univ.Press, 1993).

Disseminated tumor cells are determined from the blood samples asfollows.

1. Tumor Cell Enrichment:

25 ml of peripheral blood were centrifuged in an OncoQuick® tube(Greiner bio-one, Altmünster, Austria) at 1600×g for 20 min at 4° C. Thephase containing the tumor cells was transferred into a furthercentrifuge tube, and a cell pellet was recovered by centrifuging. Thispellet was re-suspended. The cellular portion of the suspension wascentrifuged onto a slide for a microscopic examination. The slide wasstored at −20° C. until evaluation.

2. Tumor Cell Determination

A solution containing fluorescence-labelled specific antibodies wasapplied to the slide that contained the isolated and enriched tumorcells. After an incubation period of 30 min, the tumor cells labelled bythe antibody binding were visualised under the fluorescence microscope(Axioplan Zeiss, Jena, Germany) and counted. The content of tumor cellsin the blood was calculated according to the enrichment factor. Themethod was validated with standard tumor cell suspensions.

As the labelled specific antibody, e.g. IGN311 (Anti-Lewis Y) andA45-B/B3 Anti-Cytokeratin (200 μg/ml, Micromet, Martinsried, Germany),both conjugated with fluorescent proteins, were used.

Example 2 Inhibition of the Tumor Growth by Antibodies Against Lewis-Yin Xenograft Models in Naked Mice

The antibody IGN311 (humanized IgG1, according to EP 0 528 767, e.g.)and its murine precursor ABL364 (IgG3, EP 0 547 079), respectively,recognize the Lewis Y antigen, a difucosylated lactosamine-glycosideresidue on surface molecules. In some cells, in particular in tumorcells, also receptors for growth factors are modified with Lewis Yantigen. This also applies to the family of the EGF receptors (erbB1,erbB2, erbB3, erbB4, cf. A995/2002). Therefore, it shall be examinedwith corresponding human tumor cell lines (A431) whether IGN311 alsosuppresses the establishment of tumors in vivo, in a xenograft model,inhibits the growth of an existing tumor, and prevents a disseminateddisease. By comparing the murine precursor ABL364 with IGN311, it shallalso be examined whether or not the fixing of complement componentsplays a role in the elimination of the tumor cells.

It is, i.a., an object to prove experimentally an effectiveness ofIGN311 in epithelial tumor models. This provides a prerequisite for theuse of IGN311 in the therapy of human diseases.

Material and Methods:

Material:

The fetal calf serum was obtained from PAA Laboratories (Linz, Austria),Dulbecco's modified Eagle Medium (DMEM, RPMI-1640), non-essential aminoacids, β-mercaptoethanol, were obtained from GIBCO-BRL (Grand Island,N.Y.). The A431 cell line was purchased from ATCC (Manassas, Va.).L-glutamine, penicillin G and streptomycin were obtained from SigmaChemical Co.

Cell culture: A431 cells (human epidermal cancer cell line from vulvacarcinoma) were cultured in Dulbecco's modified Eagle Medium (DMEM),containing 10% calf serum, 4 mM L-glutamine, 100 units/ml of penicillinG and 100 μg/ml of streptomycin at 5% CO₂ and 37° C. SKBR3 cells (fromhuman breast cancer) were cultured in RPMI-1640 medium at 5% CO₂ and 37°C., wherein the medium was admixed with 10% calf serum, 2 mML-glutamine, 100 units/ml of penicillin G and 100 μg/ml of streptomycin.

Keeping of animals, xenograft inoculation: pathogen-free female BALB/Cnu/nu mice, 5-6 weeks old, were used for the example (source:Versuchstierzucht (test animal husbandry) Himberg). The animals had aweight of from 18 to 20 g.

Because of their immunodeficiency, BALB/c (nu/nu) mice must be keptunder pathogen-free conditions in special filter cages (Seal-Safe-IVCcages, Techniplast, Munich). Cages with bedding, drinking water and feedwere autoclaved. Manipulations on the test animals were carried out in asterile workbench. The injected cell lines were checked forcontamination with mycoplasmas (a PCR kit being available for thispurpose). Culture supernatants of A431 cells were admixed to thesemurine cell cultures on a test basis and checked for a cytopathiceffect; if the latter occurs, it is seen as an indication of thepresence of a viral contamination. Naturally, these cells are notadministered to the naked mice before the problem of the viralcontamination has been eliminated.

Generating Different Tumors in Naked Mice

In 6 mice each per group, ˜5*10⁶ tumor cells are injected subcutaneously(s.c.) into the region of the left flank:

For this purpose, the A431 cells are re-suspended in 200 μl ofphosphate-buffered saline solution; after 6 weeks at the latest, themice are sacrificed by means of cervical dislocation and evaluated.

The administration of the antibodies is by intraperitoneal injection(twice per week). The first administration is on the next day, i.e.approximately 21 hours (24 hours at the most) after application of thetumor cells.

The animals are treated with IGN311 for 4 weeks. Subsequently, the innerorgans (in particular the lung) are examined for the formation ofmetastases. The quantification is by counting the visible lesions.

Data Analysis

The measurement parameter is the tumor size; the latter is determinedboth via the weight of the tumor (=at the end) and calculated from itsdimensions (twice per week); the latter is effected on the basis of themeasurement of the (largest) longitudinal and transverse diameter andthe thickness, according to the formula for the volume of an ellipsoid(12): volume=length*width*height*π/6, or with smaller tumors whosethickness cannot be determined with certainty: volume=length*width2*π/6.Alternatively, the weight of the tumors is determined.

Results:

At the beginning, a titration study was carried out so as to determinethe number of the tumor cells which is necessary for the inoculation.The range was between 0.5-5×10⁶ cells. Tumors were obtained with theA431 cells.

4 mice/group were inoculated with 1×10⁶ A431 cells, the administrationof the antibodies was on the next day in an amount of 10 mg/kg of IGN311or ABL364, respectively.

FIG. 1 shows the inhibition of the growth of the tumor xenograft byIGN311 and ABL364. The administration of the antibodies was startedwithin a day after the subcutaneous tumor cell application.

On day 25 the animals were sacrificed, the tumor mass was removed andits weight determined.

In a further test series, 3×10⁶ tumor cells were inoculated, which ledto a rapid tumor formation. Here it was tested whether an increase inthe IGN311 dose from 10 mg/kg to 30 mg/kg would lead to an additionalgrowth inhibition. As is apparent from FIG. 2, IGN311 shows aninhibiting activity also at an increased dose.

The determination of the tumor volume was found to be difficult sincethe tumors did not grow as an ellipsoidal body. Therefore, the animalswere sacrificed on day 16, and the weight of the tumor mass wasdetermined. The results can be taken from FIG. 3.

Based on the data obtained, it can be stated that in this xenograftmodel, IGN311 exhibits a significant anti-tumor activity in vivo andthat the effect of the IGN311 is not dramatically increased byincreasing its dose from 10 mg/kg to 30 mg/kg.

In an alternative test set-up, the proof of the inhibition of tumorgrowth and of the dissemination of the tumor cells, respectively, by theantibodies is provided in that the antibodies are administered within 24hours at the most before the tumor cells are applied subcutaneously orintravenously. Again, the tumor cells may be applied subcutaneously,intravenously or directly into the lung of the test animals.

On the live animals, the tumor growth is determined via the measurementof the tumor size, after several days or weeks the animals aresacrificed by cervical dislocation, and the tumor size is determined.Furthermore, the organs are removed and examined for micro-metastasesand other tumor-caused changes by means of various methods.

Example 3 Proof of the Reduction of Circulating EpCAM Cells in Blood

Patients, Material and Methods:

A murine IgG2a Mab having EpCAM mimick properties was used for thevaccination. The antibody was subcutaneously administered in an amountof 0.5 mg Mab, adsorbed on 1.67 mg of aluminum hydroxide in 0.5 ml ofbuffer.

As test subjects, patients suffering from cancer (more than 19 years ofage) were chosen in whom conventional therapy was unsuccessful. Thetreatment was effected with 0.5 mg of antibody subcutaneously on days 1,15, 29, 57.

Analysis of Circulating Tumor Cells in Peripheral Blood:

5 ml of heparinized blood were drawn on days 1, 29, and 71. After a milderythrocyte lysis, the residual fluid was incubated with para-magneticanti-EpCAM antibody, and the cells were separated on a magnetic column(Miltenyi). After the elution, the bound cells were labelled with FITCanti-EpCAM antibody and analysed, or counted in an inversionfluorescence microscope, respectively.

The results can be taken from FIG. 4 in which the reduction of thecirculating EpCAM cells in blood is clearly visible.

Example 4 Administration of IGN311 to Patients with Pleural Effusion

In a clinical study, 100 mg of IGN311 is administered intravenously in asingle dose to patients (from 18 to 80 years of age) who suffer fromcancer and have a pleural effusion, 24 hours at the most before removalof the effusion.

Both the volume of the pleural fluid which is removed by draining andthe presence of Lewis y positive tumor cells are determined. Thebiological activity of the IGN311 in the pleural effusion can beanalyzed by CDC determination. Moreover, it can be determined whether ornot the time until the pleurodesis can be carried out, i.e. until thepleural fluid has been drained, can be shortened by administering theantibody.

1. A method for the intra-operative treatment of a tumor to inhibitdissemination of tumor cells, which comprises administering to thepatient an antibody directed against a tumor-associated antigen duringan intra-operative treatment whereby immunocomplexing of tumor cellswithin the scope of the surgical intervention inhibits dissemination oftumor cells.
 2. The method according to claim 1, wherein the antibody isdirected against an epitope of a surface antigen of a tumor cell.
 3. Themethod according to claim 1 or 2, wherein the tumor cell is anepithelial tumor cell.
 4. The method according to claim 1, wherein theantibody is directed against an epitope of an antigen selected from thegroup consisting of peptides, proteins, carbohydrates, and glycolipids.5. The method according to claim 1, wherein the antibody is in anantibody mixture of various antibodies having a specificity fortumor-associated antigens.
 6. The method according to claim 1, whereinthe antibody functionally activates the immune system, according to anADCC and CDC effector function.
 7. The method according to claim 1,wherein the antibody binds to the tumor-associated antigen with anaffinity corresponding to a dissociation constant below a Kd value of10⁻⁶ mol/l.
 8. The method according to claim 1, characterised in thatthe antibody is derived from murine, chimeric, humanized and/or humansources.
 9. The method according to claim 1, wherein the medicament issystemically used with a single dose of at least 50 mg per patient. 10.The method according to claim 1, wherein the medicament is locallyapplied to the tumor tissue and/or to the wound area.
 11. The methodaccording to claim 1, wherein the medicament is administered immediatelyduring or before the surgical intervention.
 12. The method according toclaim 1, wherein the surgical intervention is carried out for a biopsyand/or for the removal of a solid tumor.
 13. The method according toclaim 1, wherein the surgical intervention is carried out for adetermination regarding the malignancy of a tumor.
 14. The methodaccording to claim 1, wherein the antibody is determined on theimmunocomplexed tumor tissue after the surgical intervention.
 15. Themethod according to claim 1, wherein the antibody is determined on tumorcells in blood or serum samples.
 16. A kit for the intra-operativetreatment of tumor patients, comprising a) a medicament based on anantibody directed against a tumor-associated antigen, and b) a means forthe diagnostic determination of malignant tumor cells which areimmunocomplexed with the antibody.
 17. The method according to claim 4,wherein the antigen is a member selected from the group consisting ofEpCAM, NCAM, CEA, Lews Y, Sialyl-TN, Globo H, GD2, GD3 and GM2.
 18. Themethod according to claim 7, wherein said Kd value is 10⁻⁷ mol/l. 19.The method according to claim 7, wherein said Kd value is 10⁻⁸ mol/l.20. The method according to claim 8, wherein said single does is atleast 100 mg.
 21. The method according to claim 8, wherein said singledoes is at least 200 mg.
 22. The method according to claim 8, whereinsaid single does is at most 2 mg.
 23. The method according to claim 11,wherein the medicament is administered within 24 hours before thesurgical intervention.
 24. The method according to claim 11, wherein themedicament is administered within 4 hours before the surgicalintervention.